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Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

Wojciechowski M, Rafalski D, Kucharski R, Misztal K, Maleszka J, Bochtler M, Maleszka R - Open Biol (2014)

Bottom Line: AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains.Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species.In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

ABSTRACT
In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

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Related in: MedlinePlus

(a) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam− Dcm−). All lanes are from the same TLC plate. (b) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. (c) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in (b), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.
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RSOB140110F1: (a) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam− Dcm−). All lanes are from the same TLC plate. (b) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. (c) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in (b), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.

Mentions: We have used three methods to show the presence of 5hmC in vivo. First, by using thin layer chromatography (TLC), we have confirmed that a spot at the position expected for 5hmC is detectable in DNA samples extracted from A. mellifera (figure 1a). In comparison with the mouse brain DNA, the intensity of the honeybee signal is much weaker, most likely reflecting the two orders of magnitude lower levels of CpG methylation [13,17] and hydroxymethylation (see below) in this species. Next, we have used the immunoblot assay to estimate 5hmC levels in different castes, tissues and developmental stages. As a reference, we used PCR products that were made with 5hmC nucleoside triphosphate instead of the usual cytidine triphosphate (CTP). As shown in figure 1b,c, 5hmC is detectable in all examined samples with the highest levels found in drone testes and queen ovaries. As in mammals, 5hmC levels appear to be relatively high in brain compared with other tissues. Very low levels of 5hmC are present during metamorphosis in pupae of all three castes (workers, queen, drones) and in the hypopharyngeal gland (not shown), whereas both haploid and diploid embryos show low to moderate levels of 5hmC. We also note that the variation in the amount of 5hmC in A. mellifera genomic DNA samples appears to be larger than the variation that would be expected from DNA damage. Although control experiments have established that the 5hmC antibody does not significantly cross-react with 5mC or cytosine, we cannot rule out the possibility that it detects unknown antigens other than 5hmC in our samples. Moreover, the efficiency of the antibody is known to depend on the density of 5hmC [9]. To obtain further evidence, we have conducted additional 5hmC quantifications using an alternative approach, namely the glucosyltransferase assay that is not affected by these limitations. The T4-glucosyltransferase assay depends on the transfer of radioactively labelled glucose from UDP-glucose to 5hmC. Control experiments have shown that transfer of glucose to 5-hydroxymethyluracil, a base that could be present owing to oxidative damage to thymines, is undetectable (not shown), suggesting that the assay exclusively quantifies 5hmC. The results are shown in figure 1c. From the comparison with the calibration curve, we estimate that the highest number of 5hmCs in the A. mellifera genomic samples is approximately 7000 per haploid genome (figure 1c) and thus is at least two to three orders of magnitude lower than the total number of 5hmCs per haploid genome in mammals [7,11]. The results generated using the immune blots and glucosyltransferase assay (figure 1c) are in excellent agreement with the overall correlation between the two methods, calculated to be 0.712. Furthermore, both the T4-glucosyltransferase assay and immunoblots have been conducted twice on separate biological materials collected from different colonies a few months apart yielding similar results.Figure 1.


Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

Wojciechowski M, Rafalski D, Kucharski R, Misztal K, Maleszka J, Bochtler M, Maleszka R - Open Biol (2014)

(a) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam− Dcm−). All lanes are from the same TLC plate. (b) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. (c) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in (b), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150289&req=5

RSOB140110F1: (a) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam− Dcm−). All lanes are from the same TLC plate. (b) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. (c) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in (b), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.
Mentions: We have used three methods to show the presence of 5hmC in vivo. First, by using thin layer chromatography (TLC), we have confirmed that a spot at the position expected for 5hmC is detectable in DNA samples extracted from A. mellifera (figure 1a). In comparison with the mouse brain DNA, the intensity of the honeybee signal is much weaker, most likely reflecting the two orders of magnitude lower levels of CpG methylation [13,17] and hydroxymethylation (see below) in this species. Next, we have used the immunoblot assay to estimate 5hmC levels in different castes, tissues and developmental stages. As a reference, we used PCR products that were made with 5hmC nucleoside triphosphate instead of the usual cytidine triphosphate (CTP). As shown in figure 1b,c, 5hmC is detectable in all examined samples with the highest levels found in drone testes and queen ovaries. As in mammals, 5hmC levels appear to be relatively high in brain compared with other tissues. Very low levels of 5hmC are present during metamorphosis in pupae of all three castes (workers, queen, drones) and in the hypopharyngeal gland (not shown), whereas both haploid and diploid embryos show low to moderate levels of 5hmC. We also note that the variation in the amount of 5hmC in A. mellifera genomic DNA samples appears to be larger than the variation that would be expected from DNA damage. Although control experiments have established that the 5hmC antibody does not significantly cross-react with 5mC or cytosine, we cannot rule out the possibility that it detects unknown antigens other than 5hmC in our samples. Moreover, the efficiency of the antibody is known to depend on the density of 5hmC [9]. To obtain further evidence, we have conducted additional 5hmC quantifications using an alternative approach, namely the glucosyltransferase assay that is not affected by these limitations. The T4-glucosyltransferase assay depends on the transfer of radioactively labelled glucose from UDP-glucose to 5hmC. Control experiments have shown that transfer of glucose to 5-hydroxymethyluracil, a base that could be present owing to oxidative damage to thymines, is undetectable (not shown), suggesting that the assay exclusively quantifies 5hmC. The results are shown in figure 1c. From the comparison with the calibration curve, we estimate that the highest number of 5hmCs in the A. mellifera genomic samples is approximately 7000 per haploid genome (figure 1c) and thus is at least two to three orders of magnitude lower than the total number of 5hmCs per haploid genome in mammals [7,11]. The results generated using the immune blots and glucosyltransferase assay (figure 1c) are in excellent agreement with the overall correlation between the two methods, calculated to be 0.712. Furthermore, both the T4-glucosyltransferase assay and immunoblots have been conducted twice on separate biological materials collected from different colonies a few months apart yielding similar results.Figure 1.

Bottom Line: AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains.Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species.In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

ABSTRACT
In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

Show MeSH
Related in: MedlinePlus